FI111223B - Immunotherapeutic use - Google Patents

Description

111223

This invention relates to the use of immunotherapeutic agents for the manufacture of a medicament for delaying or inhibiting the growth or spread of tumors.

It has been suggested in the art that the immune system is capable of blocking or delaying the growth of tumors, at least in some cases, and some studies seem to support this notion. Thus, Coley [W.B. 1894 "Treatment of Inoperable Malignant Injury with Toxins of Toxins of Erysipelas and the Bacillus Prodigiosus" (Treatment of Malignant Tumors That Can not be Cut with Toxins of Erysipelas and Bacillus prodigiosus) has described an immuno-therapeutic method that appears to be effective [ ), by inducing a necrotizing mechanism similar to that induced by Koch immunotherapy for tuberculosis, followed by most approaches to provide immunosuppression for cancer in some cases with the rapid progression of 20% of the tissue produced by Cooley.

It is known to have at least two maturation modes of T helper cells, referred to as TH1 and TH2. The former is associated with the formation of interleukin 2 (IL2), interferon gamma (IFN) and cytotoxic T cells (CTL), the reduction of antibody production, and the regulation of TH2 cell production. The latter produces IL4, IL5, enhances antibody production and suppresses the formation of TH1 cells. The phenomenon of Koch, which is likely to be induced by the Coley toxin, is thought to be due to a mixture of mature TH1 + TH2-v * ·· 30 cells.

It has been found that there are two different classes of immune reactivity that can be utilized to control tumors, both of which can be induced by the Bacillus Calmette-Guerin vaccine (BCG). One mechanism is 35 2,111,223 tissue destructive, including, for example, Koch and Coley immunotherapy and tumor immunotherapy with TNFα, and this mechanism is likely to involve cytokine-mediated tissue damage at sites of inflammatory K cell involvement: in the phenomenon. This mechanism appears to be effective against mesoderm-derived tumors. Another mechanism, based on the immunological identification of the tumor cells itself, may be effective against tumors of any origin. Indirect evidence of the existence of a non-specific TH1 antitumor mechanism has been derived from the successful use of recombinant interleukin 12, a potent TH1 inducer, in experimental cancers.

15 It is hypothesized that mycobacteria, when used in a manner that avoids induction of the Koch phenomenon, may initiate the identification of stressed autologous cells by epitopes between heat shock protein (HSP) epitopes and other proteins with highly unchanged sequence. selective destruction. We have come to the conclusion that the varying success of BCG immunotherapy in the past was due to a lack of understanding in the industry of BCG's efforts to enhance the already initiated response pattern, as evidenced by 25 studies of its ability to protect against tuberculosis and leprosy.

When used as a vaccine against diseases caused by mycobacteria (both tuberculosis and leprosy), BCG exhibits a wide range of effects, with protection from 80% to 0% in different studies. Therefore, it is significant *. it is contemplated that post-natal BCG generally appears to protect childhood leukemia only in those geographical locations where it also protects against tuberculosis (Grange JM, 35 Stanford JL, BCG Vaccination and Cancer. Tubercle. (1990); 71: 61-64). In other words, it protects against both conditions at sites where its response to mycobacterial antigens is not tissue necrotizing and where this response is not a Koch phenomenon.

Many tumor cells may have bacteriomimetic carbohydrate antigens and epitopes associated with the Class 1 and Class 2 High Histocompatibility Complex (MHC) on exercise proteins and other highly unchanged proteins. Under conditions that involve high antibody production, anti-carbohydrate antibodies originally developed against bacterial so-10 strains, possibly of the IgA type, may mask tumor cells and protect their surface components from cellular immune attacks.

As we have shown in our international patent application PCT / GB93 / 00463, immunotherapy with mycobacterial M. vaccae mainly induces a TH1 response pattern by activating macrophages with gamma-IFN and only low antibody production. Such TH1 responses are known to enhance CTL cell formation. The marked decrease in antibody production, coupled with the increased number of CTL cells, exposes tumor cells to be attacked by both macrophages activated after binding to bacteriomimetic blind cells on the tumor cell and by CTL cells that recognize Category 1: epitopes on the MHC complex, the stress protein, and other well-preserved proteins. It is assumed that the tissue necrotizing mechanism due to the Koch phenomenon is largely suppressed by the use of the M? 30 vaccae preparation.

As a result, immunotherapy with M. vaccae can be expected to be effective in preventing or delaying tumors that originate in meso-dermal, endoderm (viscera), and ectoderm (exogenous ketone), including breast cancer and bronchial cancer. the growth or spread of these tumors.

4, 111223

Accordingly, the present invention provides the use of antigenic and / or immune regulating materials derived from Mycobacterium vaccae for the preparation of a medicament for use in inhibiting or inhibiting the growth of tumors. This material may be administered to the subject in an amount sufficient to delay or prevent the growth or spread of the tumor.

The therapeutic agent of the invention suitably and thus preferably includes dead M. vaccae cells, most preferably cells killed by autoclaving. This immunotherapeutic agent comprises more than 108 microorganisms / ml of diluent, preferably 108 to 10 µl of killed M. vaccae microorganisms per ml of diluent. The invention also encompasses antigenic and / or immune regulatory material derived from M. vaccae which can be used in a treatment that inhibits or prevents tumor growth or spread. The essential features of the invention are set forth in the appended claims.

The diluent may be solely pyrogen-free saline or borate buffer, pH 8.0, suitable for injection.

The diluent should be sterile. A suitable borate buffer is:

Na2B4O, .10H2O 3.63g: H3B03 5.25g

NaCl 6.19 g 25 Tween 0.0005%

Distilled water to one liter.

A preferred strain of M. vaccae is a strain known as R877R, which has been isolated from mud samples from the Lango region of Central Uganda (J.L. Stanford and R.C. Paul, Ann. Soc. Beige Med.

30 Trop. , 5.3, 141-389 (1973). This strain is a persistent coarse variant and can be identified as belonging to M. vaccae by biochemical and antigenic assessments (R. Boni-cke, SE Juhasz., Zentr. Albl. Bacteriol. Parasitenkd. Infection skr. Hry. Abt. 1, Orig. ., 1964, 192, 133).

5 111223 R877R is deposited in the National Collection of Type Cultures (NCTC) Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, United Kingdom, 13 February 1984 under accession number NCTC 11659.

5 * To prepare this immunotherapeutic agent, the M. vaccae microorganism can be grown on a suitable solid medium. A modified Sauton liquid medium (S.V. Boyden and E. Sorkin, J. Immunol. 1955, 75, 15) solidified with agar is preferred. The solid support preferably contains 1.3% aga.

The medium inoculated with these microorganisms is incubated under aerobic conditions to allow these microorganisms to grow, usually at 32 ° C for 10 days. The organisms are harvested, then weighed and suspended in the diluent. This diluent may be unbuffered saline but is preferably buffered with borate and contains a surfactant such as Tween 80 as described above. The suspension is diluted to give 100 mg of microorganism / ml. The suspension is further diluted preferably with borate buffered saline so that the suspension contains 10 mg (wet weight) of microorganisms per ml of diluent. The suspension can then be dispensed into 5 ml multidose vials. Although the bottles. Thus, microorganisms can be killed by irradiation with, for example, 60cobalt irradiation at a dose of 2.5 megarads, or by any other means, e.g. 69 bar) for 10 minutes (115-125 ° C). It has been found that autoclaving produces a more effective preparation than irradiation.

This immunotherapeutic agent is generally administered by injection into the skin in a volume of 0.1 to 0.2 ml, preferably 0.1 ml. A single dose will generally contain 10 7 to 10 10 M. vaccae microorganisms killed. Patients are preferably administered a single dose of 6,111,223 containing 108-109 killed M. yaccae organisms. However, this dose may also be repeated, depending on the condition of the patient.

Although this immunotherapeutic agent is generally administered by injection into the skin, other modes of administration, such as oral administration, may be used.

The present invention also encompasses a method of delaying or inhibiting tumor growth or proliferation comprising administering to a subject afflicted with a tumor an antigen and / or immune regulatory material from Mycobacterium vaccae in an amount sufficient to elicit an effective immune response to inhibit the growth or proliferation of the tumor. prevent it.

It is preferred and according to the invention that more than one strain of M. vaccae is used. and / or that other mycobacterial components are included in this therapeutic agent. Tuberculin may also be included.

This conditioner may also include a BCG (Bacillus Calmette-Guerin) vaccine, especially freeze-dried form of the vaccine, to enhance its effect.

I f

The conditioner may contain other ingredients such as adjuvants, preservatives, stabilizers, etc. It may be provided in the form of a sterile injectable liquid or a sterile freeze-dried form which is brought into a liquid form prior to use.

The M. vaccae bacterium may be used as such, or an extract or fractionated portion of this organism may be used to prepare the therapeutic agents of the invention.

The invention is further illustrated by the following examples.

7, 111223

Example 1 M. vaccae orctanism is grown on solid medium containing 1.3% agar-solidified modified Sautonri medium. These 5 media are inoculated with microorganisms and incubated for 10 days at 32 ° C to allow the microorganisms to grow. The microorganisms are then harvested and weighed and suspended in a diluent to obtain 100 mg of microorganisms per ml of diluent. This suspension is then further diluted with buffered saline so that the resulting suspension contains 10 mg (wet weight) of microorganisms / ml of diluent, and the suspension is dispensed into 5 ml multidose vials. These vials containing these live microorganisms are then autoclaved for 10 minutes at 10 psi (about 0.69 bar) to kill the microorganisms and provide the immunotherapeutic agent of the invention, which can be (if desired) further diluted for use.

This immunotherapeutic agent may be administered by intradermal injection as described above.

Example 2

Mj_vaccae injections were given to many tumor patients.

25 Some of the results obtained are as follows: «

Breast carcinoma (l) The National Institute of Hygiene and Epidemiology, Vietnam, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, which has 30 ongoing studies using Mycobacterium vaccae for immunotherapy. A work-in-one developed breast cancer with metastases in the armpit. After the cancer had been removed by surgery, BCG immunotherapy was recommended. Because patient 35 had seen other patients undergoing such treatment, he was not enthusiastic about BCG, but requested that he be treated with M. vaccae. The patient received 109 standard injections of 8,111,223 M. vaccae, after which he has been well for at least 3 years and has not been found to have any other tumors.

2) Within a few weeks of giving an M. vaccae injection to a 50-year-old female to treat persistent dandruff disease (pityriasis), a lump was routinely mammographed on her chest. A tissue sample showed this tumor as malignant and the nodule was removed by incision. A histopathologic study revealed a clear histocytic haematopoietic hemorrhage around the armpit lymph nodes, which were free of tumors. Approximately 9 months later, dandruff disease reappeared and the patient was given a second dose of M. vaccae, which almost immediately alleviated dementia. The patient had no evidence of 15 metastases 21 months after tumor removal.

3) A woman over the age of 70 had a tumor removed several years ago. He had suffered from back pain due to arthritis for many years, and had been given M. vaccae to relieve this condition. Shortly afterwards, a spinal X-ray revealed a number of lesions, presumed to be tumor metastases, and recommended radiation therapy. Eight weeks later, he went for radiation and a repeat x-ray showed no trace of metastasis; radiation therapy was abandoned. Two and a half years, he may well not tumors had recurred.

4) A 2-day study report of a 55-year-old general practitioner was assumed to be acute abscess. An incision in the breast showed mass very rapidly growing into a carcinoma with metastases already in the armpits. The carcinoma was removed immediately and the M. vaccae injection was given immediately after discharge from the hospital. The other two injections were given every 3 to 35 months. The surgical wound healed well and after one year no evidence of metastasis was found in the patient, even though the fear was that the disease was progressing rapidly.

♦ 9 111223 5) A 50-year-old nurse who had complained of pelvic pain for one year was diagnosed with breast tumor metastases in the bone. The patient recovered well after the first M. vaccae injection, received well chemotherapy, and was given a second M. vaccae injection. The patient is fine.

Lung tumors 1) In October 1993, a 55-year-old woman developed respiratory failure, which was found to be due to a number of non-operable lung tumors. These tumors were thought to be metastases of an unknown primary tumor. The patient was given M. vaccae injection and chemotherapy and radiation therapy initiated with the consent of the cancer patient. Patient 15 survived these treatments with significantly fewer side effects and did not want another M. vaccae injection. which was suggested to him after 6 months. Ten months after his doctor's visit, his condition worsened, chemotherapy was changed, but the patient was given a poor prognosis. The second injection of M. vac-20 cae was given in early October 1994, after which the patient recovered and received two additional injections, one in December 1994 and one on March 1, 1995. Not only was this patient much better than expected, he was also effortlessly undergone 20 chemotherapy treatments.

25th Other malignancies * 1) A 30-year-old dentist with end-stage malignancy due to a malignant mixed tumor (teratoma) was given an M. vaccae injection just before he was sent home to die. He was shown signs of improvement within the first or second day, and after three weeks, the size of the visible tumors had decreased and he ate again for several weeks for the first time.

(2) An adenocarcinoma developed in the stomach of a 36-year-old woman around 35 ulcers due to infection by Helicobacter pvlori. He was found to have numerous t 10,111,223 spreading metastases despite chemotherapy. He has received the first M. vaccae inoculum. and the intention is to continue these injections every 2 months.

5 HIV-associated tumors 1) A patient with early-stage AIDS and multiple Kaposi's sarcoma was given an M. vaccae injection in November 1993. He has not developed other tumors and is already showing signs of eradication. signals. A second M. vaccae injection was given in December 1994 after the patient had developed secondary HIV-associated infections. He is to be given recurrent inoculations of M. vaccae every 2 months.

2) A patient who had almost no CD4 + cells but showed obvious signs of AIDS was given an M. vaccae injection in June 1994, and a few weeks later a small Kaposi's sarcoma of about 0 in diameter was found in his calf. 5 cm. M. vaccae injections were continued every 2 months and in March 1995 no other morbid sites or increase in size of the original morbid site were observed in the patient.

3) A 48-year-old man with multiple carcinoma colon lesions in the oropharynx was given an M. vaccae injection in June 1949, with additional injections given every 25 months. By March 1995, the tumors had stopped regenerating at all.

t

The above descriptions support the treatment of tumors with M. vac-cae. The use of M. vaccae has not been associated with any-30 side effects, and may even have mitigated the toxic effects of chemotherapy in at least two of these patients. These early practical findings support theoretical expectations and are the first evidence of valuable antitumor activity.

35 11 111223

Example 3

A preliminary experiment was performed on Balb / C mice challenged with MM3 cells of carcinoma strain of mouse mammary gland.

Three groups of 10 mice were challenged with 107 MM3 cells. After one week, 107 M vaccae were injected into the first group. the second group received 109 WL-10 vaccae and the third group served as a control. Animals were sacrificed 40 days after challenge and examined for tumor development.

Group 1 Group 2 Group 3 15 107 M. vaccaeo9 M. vaccae comparison organism organism

Advanced 165 tumors 20 p = 0.056

No tumors 985 *

P

Claims (5)

111223 1. Användning av döda Celler av Mycobacterium vaccae 20 för tillverkning av et on the basis of som som användbart för att förröja eller förhindra tillväxt eller spridning av tumörer. Use of dead cells of Mycobacterium vaccae for the manufacture of a medicament for delaying or inhibiting the growth or spread of tumors. 2. Användning enligt patentkrav 1, autoclaving the M. vaccae-cellerna avdödats genome. Use according to claim 1, characterized in that the 'M. vaccae' cells have been killed by autoclaving. 3. Användning enligt patentkrav 1 eller 2, kennettecknad av att 25 M. vaccae härletts frän stammen som deponerats hos National Collection of Type Cultures (NCTC) Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, Storbritannien, den 13 Feb 1984, med deponeringsnurnmer NCTC 11659. 111223 Use according to claim 1 or 2, characterized in that M. vaccae is obtained from a strain deposited in the National Collection of Type Cultures (NCTC) Central 10 Public Health Laboratory, Colindale Avenue, London NW9 5HT, United Kingdom, 13 February 1984 under accession number NCTC 11659. 4. Användning enligt nägot av de föregaende patentkraven, kännetecknad av att läkemedlet omfattar frän 107 account 1010 M. Vaccae-Microanalysis per dos. ν ' Use according to any one of the preceding claims, characterized in that the medicament comprises 107-1010 M. vaccae microorganisms per dose. Use according to any one of the preceding claims, characterized in that the tumor is a tumor in the chest or bronchi. 5. Användning enligt ärgot av de föregaende patentkraven, 5 kännetecknad av att tumören är en bröst- eller bronchiole.